This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We sought an in vitro primate model for serotonin neurons. Using the rhesus embryonic stem cell (ESC) line 366.4, a protocol was developed which generates a high percentage of serotonin neurons as determined by immunocytochemistry for tryptophan hydroxylase and the serotonin reuptake transporter. In addition, the ESC-derived neurons express estrogen receptor beta (ERbeta) and progesterone receptors (PR) in a manner similar to serotonin neurons in the macaque brain. We characterized physiological functions of these neurons and examined the gene expression profile during each stage of differentiation. Different combinations of mitogenic signaling molecules, specifically fibroblast growth factor 4 (FGF4) with sonic hedgehog (SHH), improved the yield of the serotonin neurons. These ESC-derived serotonin neurons have neuronal membrane properties and express specific functional serotonin reuptake transporters (SERT) and 5HT1A autoreceptors, thus making them a useful in vitro model. The feasibility of our new optimized protocol was shown by using another rhesus ESC line ORMES-22, which efficiently differentiated to serotonin neurons. In the serotonin developmental cascade, sonic hedgehog (Shh), PTCH (Shh-R), and Fev1 transcription factor expression coincided with the induction of serotonin specific marker genes during N1-selection. However, in the ESC-derived neurons, there was significant over-representation of probe sets related to cell cycle, axon guidance &dorso-ventral axis formation. This analysis suggests that the 366.4 cell line possesses cues for serotonin differentiation at early stages of differentiation, but that ESC-derived serotonin neurons are still immature.